Most inks consist of almost insoluble pigments dispersed in water or other solvents, plus additives such as preservatives, stabilizers, pH regulators, thickeners and fragrances, among others [3] [8]. Some components commonly identified in tattoo inks and permanent make up that are linked to effects on people’s health are:

As the number of tattooed people has increased, the demand for tattoo removal has also augmented [8]. For tattoo and permanent makeup removal, one of the most used procedures is the Q-switched laser. In this regard, there is a potential risk of splitting the pigments into toxic or carcinogenic fragments after exposure to the laser. This risk can also occur during exposure of the skin to normal light, such as sunbathing [20].

Several pigment by-products have been identified under experimental conditions of photochemical decomposition after sun exposure, ultraviolet (UV) radiation or laser. Some, such as 2-methyl-5-nitroaniline, 4-nitrotoluene, 2.5-dichloroaniline and 1, 4-dichlorobenzene could have toxic effects in human cells [10].

In Ref. [20] pyrolysis conditions were applied to mimic the ruby laser-induced decomposition of copperphthalocyanine in the blue pigment in tattoo inks. They applied gas chromatography-mass spectrometry coupled to headspace analysis (DHS-GC/MS) and bi-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-ToF-MS), identifying 1.2 benzene dicarbonitrile, benzonitrile, benzene and the poisonous gas hydrogen cyanide (HCN) as the main fragmentation products.

It has also been proven that red pigment Red 22 absorbs UV radiation, causing its decomposition into aromatic amines [21]. On the other hand [18], it has been indicated that PAHs in tattoo inks can absorb UV radiation, generating reactive oxygen species.

Skin reactions. Researchers have associated the metal content in tattoo ink to allergic, eczematous, lichenoid, psudolymphomatous and granulomatous reactions [9].

Ref. [22] Reports a patient with sarcoid granulomas in the area of a black tattoo and the chemical analysis of the pigment revealed high amounts of Fe (13% w/w), Co (14 μg/g) and Ni (12 μg/g). Patch tests showed a positive reaction to Ni, Co and Cd. Tattoo inks containing Al and Ti can also induce granulomatous reactions [7].

In ancient tattoos, metals such as mercury sulfide (red pigment, cinnabar), cobalt and its salts (blue), chromium (green), cadmium (yellow, red) and magnesium (violet) were identified as allergens [23]. On the other hand, it has been reported that mercury together with chromium and cobalt are contact sensitizers with various types of skin reactions in tattooed areas. In particular, red pigments that include mercury (cinnabar) can produce a delayed hypersensitivity reaction [24].

On the other hand, 80% of the allergologically most relevant red dyes in tattoo inks and permanent makeup are organic. More than 60% of them are monoazo compounds [25].

Regarding the carcinogenic potential, tattoo inks have not been evaluated by the International Agency for Research on Cancer, IARC, although some components of the inks have been classified [26].

Cadmium and its compounds are classified as carcinogenic in humans and it is considered that the critical effects in relation to the tattoo are on the bones and kidneys. In addition, nickel is classified as a carcinogen and cutaneous sensitization is considered a critical effect (Danish Environmental Protection Agency, 2012) [12]. Cobalt and Cobalt compounds, as well as Titanium dioxide, are classified in Group 2B (possibly carcinogenic to humans) (IARC, 2018).

The composition of the inks has changed and nowadays it is less likely to find metals in tattoo inks. These have been displaced by organic dyes. Currently, most inks available on the market contain polycyclic compounds or azo pigments. Some of the PAHs and PAAs released from azo pigments found in tattoo inks have been classified as carcinogenic.

Carbon black, present in black inks, is considered a potential carcinogen (group 2B) due to its high reactivity and black inks are possibly transported to the human body through the lymphatic system, together with PAHs [18] [19]. Regarding PAHs, their critical effect in relation to tattooing is the carcinogenic potential [26].

The decomposition of azo dyes in skin tissue forms PAA that are catalyzed by solar radiation, laser treatment, or pyrolysis. In this case, the carcinogenic effect is considered critical [26]. Among these compounds, anisidine is classified as a carcinogen for humans (Group 1), Nitro-o-toluidine and Chloro-o-toluidine as a probable carcinogen (Group 2A) and 3, 3-dichlorobenzidine as a possible carcinogen (Group 2B). (IARC, 2018) [27].

TableA3 of Appendix 1 shows the classification of different components found in tattoo inks, where Group 1 corresponds to carcinogen for humans, Group 2A, probable carcinogen and Group 2B, possible carcinogen.

As indicated above, some of the PAHs contained in black tattoo inks can absorb UV radiation and generate reactive oxygen species, such as singlet oxygen. This singlet oxygen damages the cellular structure by oxidation of lipids and proteins, having a cytotoxic effect [18].

On the other hand, the phenol found in tattoo inks and permanent makeup is toxic and can damage the kidneys and the central nervous system. It is also classified as teratogenic and carcinogenic [18].

In 2003 and 2008, the European Commission published guidelines on the chemical composition of tattoo inks. These recommendations were adopted by countries such as Belgium, France, Germany, the Netherlands, Spain and Sweden in modified versions. Other European countries regulate tattooing practices and the innocuousness of facilities in terms of health and hygiene [28].

The New Zealand Environmental Protection Agency also developed guidelines for tattoo and permanent makeup substances.

Meanwhile, in the United States tattoo inks are regulated as cosmetics and the pigments used in them, as colour additives. When they are considered cosmetic, a pre-market review or approval of tattoo inks is not required. On the contrary, color additives require the submission of a request to establish their innocuousness. So far, the Food and Drug Administration (FDA) has not exercised its regulatory authority for color additives in tattoo inks and their pigments, due to their historical use and the low number of adverse events reported [8].

In this way, Resolution ResAP (2008) 1 is the most detailed normative document that establishes requirements and criteria for the security of tattoos and permanent makeup. This recommends a maximum concentration of polycyclic aromatic hydrocarbons (PAH) in tattoo inks of 0.5 ppm. For the Benzo (a) pyrene, the limit is stricter, corresponding to 5 ppb. It also lists primary aromatic amines (PAA) that should not exist in tattoo inks, nor be released fromazo dyes. Regarding metals, it indicates maximum allowed concentrations of impurities in products for tattoos and permanent makeup, limits that are presented in TableA4 of Appendix 1. It highlights the limit associated with nickel which must be “as low as technically possible” due to its high allergenic potential.

In Denmark, there is no particular regulation in regards to the chemical substances used in tattoos. But there is a regularization of chemical products according to the provisions of the Danish Working Environment Service, which states that there should not be a greater amount than 0.1% of a carcinogenic element in a substance. This should also apply to tattoo inks (Chemical Substances in Tattoo Ink Survey of chemical substances in consumer products (Kortlægningafkemiskestoffer and forbrugerprodukter) no. 116, 2012) [12].

In the US, the FDA classifies tattoo inks as cosmetics [29].

In Chile, it is the MINSAL (Ministry of Health) that regulates the use of dyes used in pharmaceutical products and cosmetics (“List of Permitted Dyes in Pharmaceutical and Cosmetic Products, Supreme Decree 31”, January 13, 2012) [30]. Also, article n˚9 of Decree 304: APPROVES REGULATIONS OF TATTOO AND SIMILAR PRACTICES, 2003, provided by the MINSAL specifies that the inks used in tattoos should not have toxic components, in addition, these should be included on the list mentioned above.

Despite the high number of tattooed people, the composition of tattoo inks, their pharmacokinetics in the human body and the potential risks of inks are insufficiently known [3] [8]. Due to the above, its study and characterization acquire vital importance. In the present study we found some anomalies in the samples. Since the analysed products are almost freely imported from a world open marked, so the questions about their innocuousness aspects could affect not only to the Chilean society. Nowadays there is a huge expansion of products and services, so in many cases the regulatory organisations do not have the required information to define legal aspects linked to these new products, nor the infrastructure to control the correct application of the new norms. These guidelines may include the governmental management for import, export inspection, and certification systems for instance, tattoo inks. Also could be included the concern for giving the correct information to the common users, in order to prevent health problems.

X-ray Spectrometer. The TXRF instrument used in this study was a S2 Picofox Bruker spectrometer enclosed in an X-ray biological shield. The system includes an X-ray metal-ceramic tube with a molybdenum target operating at 50 W of maximum power, at 50 kV and 1 mA. The system is air-cooled; and includes a multilayer monochromator; a Peltier-cooled high resolution X-Flash silicon drift detector (without liquid nitrogen cooling) with a 30 mm² active area and an energy resolution less than 140 eV at 100 kcps (Mn Kα line, 5.895 keV). The instrument was controlled by a computer by its Spectra 6.3 software.

For the calibration of the TXRF spectrometer, eight MERCK ICP single element standard solutions of S, K, Sc, V, Mn, Co, Cu and Ga were used. For the sample preparation as well as for the dilution of the samples, pure water was obtained from a feeder 55 WG with subsequent de-ionization by a Milli-Q SP Reagent Water System (Millipore) which yields ultrapure water with specific resistivity of 18 MΩ·cm. For the ink samples acid digestion MERCK concentrated supra-pure HNO₃ mineral acid was used.

For the determination of elemental X-ray line sensitivities for S, K, Sc, V, Mn, Co and Cu, two Multi-Element Solution (MES) standards having elemental concentration of 5 and 10 μg/mL were prepared by mixing the corresponding MERCK single element standards and diluting the resultant solution mixture to the required concentrations, using ~1M nitric acid. Galium was added as internal standard in these solutions with a constant concentration of 5 μg/mL. The TXRF spectra of these standards were measured for 500 s and the relative sensitivities with respect to Ga were determined, as is described in our previous studies [33] [34].

We have analyzed 48 different tattoo inks, which were acquired freely from on line suppliers, from Concepcion City, Chile. The samples can be classified in addition to colour in three different types, water based, oily based and alcohol based tattoo inks.

The 15% of these samples were digested with MERCK concentrated supra-pure HNO₃ mineral acid. All of these samples were digested utilizing 5 mL concentrated HNO₃ mineral acid, in a hot plate at 75˚C, for about 24 hours, in order to completely transfer the analytes to the homogeneous liquid samples. Vanadium was added as internal standard in these solutions with a constant concentration of 5 μg/mL. For the preparation of the blank samples, all the above steps were followed except that milli-Q water was taken in place of the tattoo inks at the beginning of the process.

The 100% of the samples were analyzed by external standard quantification. Samples were homogenized by mechanical agitation at least for 5 minutes, then the specimens were obtained from 2 μL of a single droplet. Later a 2 μL of a droplet of Ga at 5 μg/mL was added directly over to the sample holder. The very-small volumes of the droplets were chosen in order to reduce effects over the morphology of the sample [35] and effects over the excitation source [36]. We replicated the methodology previously applied for Cobalt evaluation in injectable multivitamins [37], obtaining in this case uncertainties in the order of 10%. A flow chart for the ink samples preparation is depicted in Figure 1.

The residues of the samples were obtained by heating. In all cases the TXRF spectra were acquired for 500 s. In all measurements, the dead time of the acquisition was controlled, to be in the range 3% - 6%. A detailed energy calibration of the spectrometer was made after 20 measurements.

The sources that affect the quality in a TXRF evaluation are described in the Figure 2, as is described in Ref. [38], where also are included sources influencing the right function of the electronic chain of detection. In this category we can mention the dead time, the correct energy calibration of the spectrum, and also electronic settings could be included, like a correct poles/zero calibration, etc. In particular, the impact of the dead time over a TXRF (or any spectroscopic) measurement is very difficult to evaluate, from empirical methods or by mathematical theoretical formulations.